The inner ear is a complex organ that provides us with the ability to hear and maintain balance. The development and morphogenesis of the inner ear depends upon precise temporal and spatial gene expression patterns, cell signaling and cell migration. Disruption of this regulation can lead to loss of hearing, which is common in the human population. Two or three out of every 1,000 children in the United States are born deaf or hard-of-hearing. More lose their hearing later during childhood and approximately one-third of people older than 60, and half older than 85, have hearing loss. Sensory neurons in the inner ear express a variety of microRNAs (miRs), including a three miR cluster comprised of miR-96, miR-182 and miR-183. Mutations in the seed sequence of miR-96 have been shown to result in hearing defects. A small number of inner ear target genes have been identified; however our knowledge on the overall role of these miRs and sensory neuron specification, development and maintenance is largely unknown. We have found that the sensory neurons of the ascidian larva express a transgene reporter for the ascidian miR-96, miR-182 and miR-183 cluster. Because animal sensory neurons are derived from a common ancestral cell type, ascidian sensory neurons likely share numerous sensory miR target genes with vertebrates. Ascidians are the sister group of vertebrates, but have small genomes and simplified gene regulatory networks making them ideal for regulatory analysis. We have developed the ascidian embryo as a platform for identifying and characterizing microRNAs and their gene targets. Here we will characterize the miR-96, -182 and -183 cluster and investigate conserved and unique target genes in ascidian embryos. The Specific Aims of this proposal are to: 1) Characterize expression of the sensory miR cluster in Ciona embryos. In situ hybridization with locked nucleic acid (LNA) probes, miR sponges, and ectopic transcription factor expression will be used to examine miR expression. 2) Identify gene targets regulated by the sensory miR cluster. Sensory miR gene targets shared between ascidians and humans will be assayed in vivo using a transgenic sensory assay. This research addresses fundamental questions of miR target gene regulation and will help promote ascidians as a model organism for comparative regulatory studies not only as related to hearing loss, but to other diseases as well. In addition, this research may provide novel potential targets for the treatment of sensory neuronal diseases, i.e. deafness, which are associated with SMC targets.